Abstract Atrial fibrillation (AF) is the most common sustained arrhythmia and causes significant morbidity and increased mortality. Current therapies to treat AF are inadequate and do not target the underlying mechanisms that promote, initiate or maintain arrhythmiogenesis. Activated Ca2+/calmodulin- dependent protein kinase II (CaMKII) is proarrythmic, but the specific sites of CaMKII activity crucial for AF are unknown. Mitochondrial populations of CaMKII signal to the mitochondrial Ca2+ uniporter (MCU) to increase Ca2+ entry into mitochondria, which leads to increases in mitochondrial ROS (mtROS). Sustained mtROS elevations lead to global ROS increases which promote arrhythmias, but the relative importance of mtROS in AF is unclear. In preliminary studies, I found that mitochondrial antioxidant (mitoTEMPO) treatment protects mice from AF. Based on this finding, I hypothesize that mtROS are key in AF and, moreover, that mitochondria act as proarrhythmic, ROS-generating circuits in AF following mitochondrial CaMKII activation and mitochondrial Ca2+ loading. CaMKII is ROS sensitive and can be oxidized to an autonomously active form (ox-CaMKII), suggesting that mitochondrial CaMKII may be the key switch for maintaining a feed-forward mtROS circuit. To assess this question, I will utilize mice with heart-specific mitochondrial CaMKII inhibition (mtCaMKIIN mice) to discover if they have less mtROS, less mitochondrial Ca2+ loading and protection from AF. I hypothesize that MCU- mediated mitochondrial Ca2+ loading promotes mtROS and mitochondrial CaMKII activity, and thus represents an additional potential target for preventing AF. I will use transgenic mice with a dominant negative MCU (DN-MCU mice) to establish if blocking MCU-mediated Ca2+ entry protects from mtROS increases and AF. To my knowledge, this will be the first in vivo study to determine the importance of mtROS in AF and the first study to directly test a molecular mechanism for feed-forward mtROS production in the heart. The goal of this work is to establish how mitochondrial CaMKII and mitochondrial Ca2+ contribute to mtROS production in AF and to determine whether these represent viable targets for future therapeutic development. My studies will test the hypothesized role of mtROS, mitochondrial CaMKII and MCU-mediated Ca2+ entry through two Specific Aims: Aim 1: Determine if excessive mitochondrial ox-CaMKII and mtROS are essential components of a proarrhythmic circuit for AF Aim 2: Test if genetic MCU inhibition protects against AF